EP0867190B1 - Conjugues des cytotoxines comprenant des dipeptides - Google Patents

Conjugues des cytotoxines comprenant des dipeptides Download PDF

Info

Publication number
EP0867190B1
EP0867190B1 EP96913722A EP96913722A EP0867190B1 EP 0867190 B1 EP0867190 B1 EP 0867190B1 EP 96913722 A EP96913722 A EP 96913722A EP 96913722 A EP96913722 A EP 96913722A EP 0867190 B1 EP0867190 B1 EP 0867190B1
Authority
EP
European Patent Office
Prior art keywords
compound
added
peg
solvent
pro
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP96913722A
Other languages
German (de)
English (en)
Other versions
EP0867190A1 (fr
EP0867190A4 (fr
Inventor
Toshiyuki Suzawa
Motoo Yamasaki
Satoru Nagamura
Hiromitsu Saito
So Ohta
Nobuo Hanai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
KH Neochem Co Ltd
Original Assignee
Kyowa Hakko Kogyo Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Hakko Kogyo Co Ltd filed Critical Kyowa Hakko Kogyo Co Ltd
Publication of EP0867190A1 publication Critical patent/EP0867190A1/fr
Publication of EP0867190A4 publication Critical patent/EP0867190A4/fr
Application granted granted Critical
Publication of EP0867190B1 publication Critical patent/EP0867190B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/06—Peptides being immobilised on, or in, an organic carrier attached to the carrier via a bridging agent

Definitions

  • Cytotoxin conjugates in which a cytotoxin is bound through a spacer to a residue derived from a compound which has an affinity for a target cell, for example, a residue derived from an antibody or antibody fragment which is specific to a cancer are described.
  • the cytotoxin conjugate obtained inhibits the growth of a target cell selectively and efficiently, and is useful as an active ingredient of an antitumor agent.
  • Anthracycline anticancer compounds so far known include daunomycin ( US Patent No. 3,590,028 ) and adriamycin ( US Patent No. 3,590,028 ), which are in wide clinical use as anticancer agents.
  • side effects of these compounds have been reported; for example, adriamycin is known to have side effects such as cardial toxicity and marrow depression [Cancer Chemotherapy and Pharmacology, 4 , 5-10 (1980)]. Alleviation of such side effects is a big problem to be solved, and comprehensive research has so far been made to this end. Specifically, in recent years, research on drug delivery systems has been pursued aiming at alleviation of toxicity, maintenance of concentration in blood and improvement of affinity for a cancer cell.
  • antibody conjugates having a specificity to a cancer cell have been studied. Some examples of such conjugates are shown below [ Bioconjugate Chem., 1, 13 (1990 )]. stracture cytotoxin vinblastine risin A diphtheria toxin A abrin A vinblastine hydrazide methotrexate hydrazide anthracycline chelates of indium and yttrium metal chelates anthracycline
  • US5219564 describes copolymers of a poly-(alkene oxide) and an amino acid or peptide sequence having multiple pendent functional groups at regular predetermined intervals that can be utilised for drug attachment or cross-linking reactions.
  • WO 92/20371 describes a conjugate of bioactive compounds comprising a synthetic hydrophilic polymer spacer such as PEG.
  • WHO 92/16221 describes conjugate of bioactive compounds comprising a non-peptidic polymeric spacer such as PEG.
  • the present inventors made intensive studies in search of an excellent cytotoxin conjugate which kills tumor cells selectively. As a result, the inventors have found that a conjugate having a spacer which is specifically cleaved when introduced into a specific cell can be obtained by chemically binding a cytotoxin to a compound which has a specific affinity for a cancer cell through a novel spacer comprising polyethylene glycol and dipeptide. Thus the present invention has been completed.
  • the present invention relates to a cytotoxin conjugate in which a residue derived from a compound having an affinity for a target cell is bound to a cytotoxin through a spacer comprising polyalkylene glycol and dipeptide.
  • Typical examples of the conjugates of the present invention are cytotoxin conjugates represented by general formula (A): Z( ⁇ X 1 -CH 2 (OCH 2 CH 2 ) n OCH 2 CO-R 1 -R 2 -W-Y 1 ) m (A) wherein Z represents a residue derived from a compound having an affinity for a target cell; Y 1 represents a cytotoxin; R 1 and R 2, which may be the same or different, each represents an amino acid residue; Alk represents alkylene; n represents an integer of 1-1000; and m represents an integer of 1-100.
  • X 0 , W 0 and W 1 are not specifically defined, examples of their representations are as follows:
  • Alk 1 and Alk 2 which may be the same or different, each represents a straight-chain or branched alkylene having 1-8 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, heptylene and octylene.
  • cytotoxin conjugates are compounds represented by general formula (I): Z( ⁇ X 1 -CH 2 (OCH 2 CH 2 ) n OCH 2 CO-R 1 -R 2 -W-Y 1 ) m (I) wherein X 1 represents CO, S or
  • W represents a single bond or and Z, Y 1 , R 1 , R 2 , n and m have the same meanings as defined above.
  • the compounds represented by general formula (I) are hereinafter referred to as Compounds (I), and the same applies to the compounds of other formula numbers.
  • the alkylene moiety of the alkylene and the polyalkylene glycol means a straight-chain or branched alkylene having 1-8 carbon atoms such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, heptylene, and octylene.
  • Examples of the compounds which have an affinity for a target cell are compounds having a structure capable of binding to X 1 such as COOH, NH, SH, and OH, e.g., receptor ligands such as epidermal growth factors (EGF) and transferrin having an affinity for a target cell, adhesion molecules represented by the arginine-glycine-aspartic acid sequence, and proteins and peptides such as antibodies and antibody fragments.
  • Preferred examples are antibodies and antibody fragments.
  • the antibodies include polyclonal antibodies and monoclonal antibodies produced according to known methods which belong to immunoglobulin (Ig) classes such as IgG, IgA, IgM, and IgE, and immunoglobulin subclasses, for example, IgG 1 , IgG 2 , IgG 3 , and IgG 4 in the case of IgG.
  • Ig immunoglobulin
  • Preferred examples are KM-641 antibody which is an antibody against ganglioside GD 3 which is highly expressed in a cancer cell ( Japanese Published Unexamined Patent Application No. 176791/93 ), KM-231 (AMC-462) antibody which is an antibody against sialyl Lewis a ( Japanese Published Unexamined Patent Application No.
  • NL-1 antibody which is an antibody against common human acute lymphatic leukemia cell antigen (CALLA) [ Proc. Natl. Acad. Sci. USA 79, 4386-4390 (1982 )].
  • CALLA common human acute lymphatic leukemia cell antigen
  • the antibody fragments are F(ab') 2 obtained by treating the above-mentioned antibodies with a proteolytic enzyme such as pepsin, Fab' obtained by reducing F(ab') 2 with mercaptan, and Fab obtained by degrading the antibodies with a proteolytic enzyme such as papain, trypsin, chymotrypsin, and plasmin.
  • cytotoxins having a structure capable of condensing with a carboxyl group of the terminal amino acid R 2 or capable of attaching to a double bond of maleinimide, such as NH, SH and OH, e.g., anthracycline compounds such as adriamycin ( US Patent No. 3,590,028 ) and daunorubicin ( US Patent No.
  • duocarmycin derivatives such as DC-88A derivatives ( Japanese Published Unexamined Patent Application No. 288879/90 ) and the compounds described in Reference Examples, mitomycin A, mitomycin C, and protein cytotoxins such as ricin A, diphtheria toxin, and Pseudomonas exotoxin.
  • the amino acid residues are an alanine residue, a leucine residue, a glycine residue, a proline residue and a valine residue.
  • the abbreviations used herein have the following meanings, unless otherwise specified.
  • the abbreviations for amino acids and their protecting groups follow the recommendations by IUPAC-IUB Joint Commission on Biochemical Nomenclature [ Biochemistry, 11, 1726 (1972 )].
  • Process 1 Process for preparing Compound (Ia), i.e., Compound (I) wherein Z is a group having N, S or O, X 1 is CO, and W is a single bond
  • Compound (Ia) can be prepared according to the following reaction steps.
  • a 1 and A 2 which may be the same or different, each represents a carboxylic acid protecting group; A 3 and A 4 , which may be the same or different, each represents a carbxylic acid activating group; Hal represents halogen; Z 1 represents a group having N, S or O in the definition of Z; and Y 1 , R 1 , R 2 and n have the same meanings as defined above.
  • Examples of the carboxylic acid protecting group are carboxylic acid protecting groups used in ordinary peptide synthesis (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen) such as tBu, Bzl, and Pic.
  • An example of the carboxylic acid activating group is ONSu.
  • the halogen means a chlorine atom, a bromine atom or an iodine atom.
  • Compound (V) can be obtained by reaction of polyethylene glycol dicarboxylic acid (III) with Compound (IV) in an amount of 0.1 to 1 equivalent, preferably, 0.5 equivalent in a solvent such as DMF in the presence of a base such as potassium carbonate at -50 to 30°C for 1 to 24 hours.
  • Diester and unreactive dicarboxylic acid contained in the obtained product can be removed by partition column chromatography, column chromatography using adsorption resins, reversed-phase silica gel, alumina, diatomaceous earth, or ion-exchange resins, preferably, silica gel column chromatography or thin layer chromatography.
  • Compound (VII) can be obtained by condensing Compound (V) with Compound (VI) obtained according to an ordinary liquid-phase peptide synthesis method (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen) in a solvent in the presence of a base in an amount of 1 to 2 equivalents, using a condensing agent in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents.
  • the base are triethylamine and NMM
  • examples of the condensing agent are ordinary amino acid condensing reagents such as DCC and EDC
  • examples of the solvent are methylene chloride, chloroform and DMF.
  • a 2 used as the carboxylic acid protecting group of Compound (VI) is a group which can be selectively removed separately from A 1 of Compound (V).
  • Compound (VII) can also be obtained by condensing Compound (V) with HONSu, HOBt, or the like in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents in a solvent in the presence of an equivalent amount of a base, using a condensing agent in an amount of 1 to 5 equivalents, preferably 1 to 2 equivalents to obtain an active ester, and then by subjecting the obtained ester to reaction with Compound (VI) at 0 to 30°C for 1 to 24 hours.
  • the base condensing agent and solvent, those which are described above can be used.
  • Compound (VIII) can be obtained by selectively removing the protecting group A 2 from Compound (VII) according to a method for the removal of a protecting group used in ordinary peptide synthesis (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen).
  • Compound (X) can be obtained by condensing Compound (VIII) with an equivalent amount of a cytotoxin in a solvent in the presence of a base in an amount of 1 to 2 equivalents, using a condensing agent in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents.
  • a base is triethylamine and NMM
  • examples of the condensing agent are ordinary amino acid condensing reagents such as DCC and EDC
  • examples of the solvent are methylene chloride, chloroform and DMF.
  • the reaction is carried out by stirring at -30 to 30°C for 1 to 24 hours.
  • Compound (X) can also be obtained by condensing Compound (VIII) with HONSu, HOBt, or the like in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents in a solvent in the presence of an equivalent amount of a base, using a condensing agent in an amount of 1 to 5 equivalents, preferably 1 to 2 equivalents to obtain an active ester (IX), and then by subjecting the obtained ester to reaction with a cytotoxin at -30 to 30°C for 1 to 24 hours.
  • a condensing agent and solvent those which are described above can be used.
  • Compound (XI) can be obtained by removing the protecting group A 1 from Compound (X) according to a method for the removal of a protecting group used in ordinary peptide synthesis (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen).
  • a protecting group used in ordinary peptide synthesis Frundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen.
  • deprotection is carried out according to ordinary methods for selectively removing amino acid protecting groups such as hydration in the presence of a palladium carbon catalyst for A 1 , and trifluoroacetic acid treatment for A 2 , whereby A 1 and A 2 can be selectively removed in each step.
  • Compound (XI) can also be obtained by removing the protecting group A 1 from Compound (IX) obtained in Step 4 according to the method of Step 5, and then subjecting the obtained compound to reaction with a cytotoxin according to the method of Step 2.
  • Compound (Ia) can be obtained from Compound (XI) and a compound which has an affinity for a target cell and has NH, SH, or OH in the molecule according to the method of Step 2.
  • the compounds having an affinity for a target cell such as proteins and peptides, are liable to be denatured and inactivated in an organic solvent, and it is preferred to carry out the above reaction under mild conditions, e.g. in an aqueous solution.
  • the reaction is carried out by dissolving a compound having an affinity for a target cell in a buffer such as a phosphate buffer or a borate buffer (pH 6-8), and adding to the solution Compound (XI) in an amount of 1 to 500 equivalents, preferably, 1 to 50 equivalents, and a condensing agent such as EDC, followed by stirring at 0 to 30°C for 1 to 48 hours.
  • a buffer such as a phosphate buffer or a borate buffer (pH 6-8)
  • a condensing agent such as EDC
  • the reaction may be carried out by obtaining an active ester (XII) according to the method of Step 2, and adding to a solution of a compound having an affinity for a target cell in a buffer (pH 6-8) the obtained active ester in an amount of 1 to 500 equivalents, preferably 1 to 50 equivalents in the presence of 0 to 10%, preferably 0 to 5% DMSO or DMF, followed by stirring at 0 to 30°C for 1 to 48 hours.
  • Process 2 Process for preparing Compound (Ib), i.e., Compound (I) wherein Z is a group having N, S or O, X 1 is CO, and W is
  • Compound (Ib) can be prepared according to the following reaction steps.
  • Compound (XIII) can be obtained from Compound (VIII) and aminoethyl maleimide according to the method of Step 2.
  • Compound (XIV) can be obtained by subjecting Compound (XIII) to reaction with a cytotoxin.
  • the reaction is carried out by dissolving a cytotoxin in a buffer such as a phosphate buffer and a borate buffer (pH 6-8), and adding Compound (XIII) in an amount of 1 to 50 equivalents to the solution, followed by stirring at 0 to 30°C for 1 to 48 hours.
  • a buffer such as a phosphate buffer and a borate buffer (pH 6-8)
  • Compound (Ib) can be obtained from Compound (XIV) according to the methods of Steps 5 and 6.
  • Process 3 Process for preparing Compound (Ic), i.e., Compound (I) wherein Z is a group having CO, X 1 is S, and W is a single bond
  • Compound (Ic) can be prepared according to the following reaction steps.
  • a 4 represents a thiol protecting group
  • Z 2 represents a group having CO in the definition of Z
  • a 1 , Y 1 , R 1 , R 2 and n have the same meanings as defined above.
  • Examples of the thiol protecting group are benzyl, picolyl, and nitrobenzyl.
  • the starting compound (XV) can be obtained according to the method for the synthesis of polyethylene glycol derivatives described in Poly (Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications (J. M. Harris, Ed., Plenum, NY. 1992).
  • Compound (XVI) can be obtained by protecting the thiol group of Compound (XV) according to a method for introducing a protecting group used in ordinary peptide synthesis (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen).
  • Compound (XVII) can be obtained from Compound (XVI) according to the method of Step 3.
  • Compound (XVIII) can be obtained from Compound (XVII) according to the methods of Steps 2 and 3.
  • Compound (XIX) can be obtained from Compound (XVIII) according to the method of Step 4.
  • Compound (XX) can be obtained by deprotecting Compound (XIX) according to a method for removing a protecting group used in ordinary peptide synthesis (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen).
  • Compound (Ic) can be obtained by binding Compound (XX) to a compound having an affinity for a target cell and having COOH in the molecule by a method such as the activation of a thiol group described in J. Applied Biochem., 6, 56-63 (1984 ).
  • Process 4 Process for preparing Compound (Id), i.e., Compound (I) wherein Z is a group having N, S or O, X 1 is and W is a single bond, and Compound (IId), i.e., Compound (II) wherein X 2 is and Y 2 is hydroxyl.
  • Compound (Id) and Compound (IId) can be prepared according to the following reaction steps. (In the formulae, A 1 , Y 1 , Z 1 , R 1 , R 2 and n have the same meanings as defined above.)
  • the starting compound (XXI) can be obtained according to the method for the synthesis of polyethylene glycol derivatives described in Poly (Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications (J. M. Harris, Ed., Plenum, NY. 1992 ).
  • Compound (XXII) can be obtained from Compound (XXI) according to the method of Step 3.
  • Compound (IId) can be obtained from Compound (XXII) according to the methods of Steps 2 and 3.
  • Compound (XXIII) can be obtained from Compound (IId) according to the method of Step 4.
  • Compound (Id) can be obtained from Compound (XXIII) and a compound having an affinity for a target cell and having NH, SH or OH in the molecule according to the method of Step 8.
  • Process 5 Process for preparing Compound (IIa), i.e., Compound (II) wherein X 2 is carboxyl and Y 2 is hydroxyl
  • Compound (IIa) can be obtained from Compound (VIII) according to the method of Step 3.
  • Process 6 Process for preparing Compound (IIb), i.e., Compound (II) wherein X 2 is carboxyl and Y 2 is
  • Compound (IIb) can be obtained from Compound (XIII) according to the method of Step 3.
  • Process 7 Process for preparing Compound (IIc), i.e., Compound (II) wherein X 2 is mercapto and Y 2 is hydroxyl
  • Compound (IIc) can be obtained from Compound (XVIII) according to the method of Step 14.
  • Compounds (I) and (II) having the desired groups at the desired positions can be obtained by combining the above-described methods appropriately.
  • the intermediates and desired compounds in the above-described processes can be isolated and purified by purification methods such as filtration, extraction, washing, drying, concentration, recrystallization, various kinds of column chromatography, e.g. silica gel chromatography, ion-exchange chromatography, reversed-phase chromatography, and gel filtration chromatography, and dialysis using an ordinary semipermeable membrane.
  • the intermediates can be subjected to the subsequent reaction without a specific purification treatment. Examples of cytotoxin Conjugates (I) obtained by the above-described processes are shown in Table 1. Table 1 Compound No.
  • Cervical cancer HeLaS3 cells (CALLA - ) having no expression of CALLA antigen and Burkitt lymphoma Daudi cells (CALLA + ) having an expression of CALLA antigen were used as target cell lines.
  • Each of the target cell suspensions was put into wells of a 96-well flat plate in an amount of 50 ⁇ l (1 x 10 3 cells/well), and cultured in a CO 2 - incubator at 37°C for 2 hours.
  • Compound (Ia-3) and Compound (Ia-1) exhibited a little inhibitory effect on the growth of HeLaS3 cells at high concentrations, whereas they exhibited a remarkable inhibitory effect on the growth of Daudi cells even at very low concentrations.
  • Compound (Ia-9) or Compound (Ia-7) was added, an inhibitory effect was hardly observed on the growth of HeLaS3 cells, while a more specific inhibitory effect was observed on the growth of Daudi cells.
  • Addition of the monoclonal antibody (NL-1) alone had little effect on the cell growth (refer to Fig. 1).
  • the inhibitory effect of Compound (Ia-10) on cell growth was examined. Cervical cancer HeLaS3 cells (sLe a- ) having no expression of sLe a antigen and large intestine cancer SW1116 cells (sLe a+ ) having an expression of sLe a antigen were used as target cell lines. Each of the target cell suspensions was put into wells of a 96-well flat plate in an amount of 50 ⁇ l (1 x 10 3 cells/well), and cultured in a CO 2 -incubator at 37°C for 2 hours.
  • Human myeloma SK-Ly-18 cells were suspended in RPMI-1640 medium containing 10% fetal calf serum at a concentration of 2 x 10 8 cells/ml, and the suspension was mixed with Matrigel (registered trademark; Becton Dickinson Labware, USA) in the ratio of 1:1 (v/v).
  • the mixture (0.1 ml, 1 x 10 7 cells/mouse) was subcutaneously transplanted into BALB/C nu/nu mice (Clea Japan, Inc.).
  • a drug-monoclonal antibody conjugate (amount corresponding to 7.5 mg/kg ADM) or ADM (7.5 mg/kg) was intravenously administered to the mice divided in groups each consisting of 5.
  • Tumor Volume mm 3 a ⁇ b 2 / 2 a: major axis (mm) b: minor axis (mm)
  • the therapeutic effect on the transplanted tumor was evaluated in terms of V/V 0 , the ratio of the tumor volume on the day of evaluation (V) to that on the day of drug administration (V 0 ).
  • V the ratio of the tumor volume on the day of evaluation
  • V 0 the ratio of the tumor volume on the day of drug administration
  • a significant tumor growth was observed in the control group, and a remarkable tumor growth inhibiting effect was observed in Compound (Ia-1)- and Compound (Ia-3)-administered groups.
  • a growth inhibiting effect was not observed in the test group to which the same quantity of ADM alone was given compared with the control group.
  • the utility of the drug-monoclonal antibody conjugates was thus demonstrated (refer to Fig. 2).
  • Fig. 1 shows the cell growth inhibiting effect of cytotoxin conjugates and a monoclonal antibody.
  • Fig. 2 shows the therapeutic effect of cytotoxin conjugates and a drug on transplanted tumor.
  • the residue was dissolved in 70 ⁇ l of DMSO, and 1400 ⁇ l of a phosphate buffer was added thereto. To the resulting mixture was added 480 ⁇ l of an aqueous solution of NL-1 antibody (3.3 mg/ml), followed by gentle stirring at 4°C for 24 hours. After the insoluble matter was removed with a filter (0.45 ⁇ m), the antibody fraction was purified by gel filtration HPLC [column: Superose 12 (Pharmacia Fine Chemicals, Inc.), developer: phosphate buffer, flow rate: 0.5 ml/min, detection: at the absorbance of 280 nm].
  • the desired fraction eluted was concentrated using a small-size ultrafiltration membrane (Millipore Corp., cut-off molecular weight: 5000) to give 771 ⁇ g of NL-1-(PEG-Ala-Val-ADM) m (protein content: 0.67 mg/ml) (yield: 49%).
  • the antibody fraction was purified by gel filtration HPLC and concentrated in the same manner as in Example 1 to give 1060 ⁇ g of NL-1-(PEG-Ala-Pro-ADM) m (protein content: 0.88 mg/ml) (yield: 63%).
  • the number of molecules of adriamycin bound was 1.8 per antibody molecule as calculated from the absorbances at 280 nm and 495 nm in the same manner as in Example 1. It was confirmed that the affinity of the conjugate was approximately equal to that of an unbound antibody according to the fluorescent antibody method described in Example 1.
  • the number of molecules of adriamycin bound was 1.5 per antibody molecule as calculated from the absorbances at 280 nm and 495 nm in the same manner as in Example 1. It was confirmed that the affinity of the conjugate was approximately equal to that of an unbound antibody according to the fluorescent antibody method described in Example 1.
  • the number of molecules of daunorubicin bound was 1.9 per antibody molecule as calculated from the absorbances at 280 nm and 495 nm in the same manner as in Example 4. It was confirmed that the affinity of the conjugate was approximately equal to that of an unbound antibody according to the fluorescent antibody method described in Example 1, wherein SW1116 cells were used as the cells for evaluation.
  • the number of molecules of daunorubicin bound was 1.5 per antibody molecule as calculated from the absorbances at 280 nm and 495 nm in the same manner as in Example 4. It was confirmed that the affinity of the conjugate was approximately equal to that of an unbound antibody according to the fluorescent antibody method described in Example 5.
  • the number of molecules of Compound (20) bound per antibody molecule was calculated by subjecting the conjugate to enzyme treatment (proline endopeptidase), and quantitatively determining released Compound (20) by HPLC according to the method described in Reference Example 27. It was found that in the obtained conjugate, the number of molecules of Compound (20) was 0.49 per antibody molecule.
  • the obtained compound (0.14 mg) was dissolved in 250 ⁇ l of a solution of HONSu in methylene chloride (0.076 mg/ml) under ice cooling, and 250 ⁇ l of a solution of DCC in methylene chloride (0.14 mg/ml) was added thereto, followed by stirring for 2.5 hours under ice cooling.
  • the insoluble matter (DCU) was removed by filtration, and the solvent was removed from the filtrate under reduced pressure at a temperature below 0°C.
  • the residue was dissolved in 36 ⁇ l of DMSO, and 204 ⁇ l of an ice-cooled phosphate buffer was added thereto.
  • the obtained compound (0.05 mg) was dissolved in 250 ⁇ l of a solution of HONSu in methylene chloride (0.028 mg/ml) under ice cooling, and 250 ⁇ l of a solution of DCC in methylene chloride (0.05 mg/ml) was added thereto, followed by stirring for 4.5 hours under ice cooling.
  • the insoluble matter (DCU) was removed by filtration, and the solvent was removed from the filtrate under reduced pressure at a temperature below 0°C.
  • the residue was dissolved in 15 ⁇ l of DMSO, and 80 ⁇ l of a cooled phosphate buffer was added thereto.
  • the obtained compound (1.47 g) was dissolved in 30 ml of THF and 30 ml of methanol, and 250 mg of 10% palladium carbon catalyst was added thereto, followed by vigorous stirring in an atmosphere of hydrogen at room temperature for 8 hours. After the catalyst was removed by filtration, the solvent was removed from the filtrate under reduced pressure.
  • the insoluble matter (DCU) was removed by filtration, and the solvent was removed from the filtrate under reduced pressure to obtain 0.21 g of a residue containing BzlO-PEG-Ala-Pro-OtBu.
  • the obtained residue was dissolved in 5.1 ml of methylene chloride, and 5.1 ml of TFA was added thereto, followed by stirring at room temperature for 24 hours.
  • the residue was subjected to purification using 20 ml of silica gel (Wako Gel C-200), and as the developer, 50 ml each of chloroform-methanol mixtures (100:0, 100:1, 50:1, 30:1, 10:1, 5:1, 3:1). The eluate was taken in 5 ml fractions.
  • the insoluble matter (DCU) was removed by filtration, and the solvent was removed from the filtrate under reduced pressure. Then the residue was subjected to purification using 50 ml of silica gel (Wako Gel C-200), and as the developer, 50 ml each of chloroform-methanol mixtures (100:0, 100:1, 50:1, 30:1, 20:1, 10:1). The eluate was taken in 5 ml fractions.
  • the insoluble matter (DCU) was removed by filtration, and the solvent was removed from the filtrate under reduced pressure. Then the residue was subjected to purification using 50 ml of silica gel (Wako Gel C-200), and as the developer, 50 ml each of chloroform-methanol mixtures (100:0, 100:1, 50:1, 30:1, 20:1). The eluate was taken in 5 ml fractions.
  • the desired HO-PEG-Ala-Val-ADM was eluted with 20 ml of a mixture of chloroform:methanol:water (13:6:1). The solvent was removed from the desired fraction under reduced pressure to give 179 ⁇ g (0.20 ⁇ mol) of HO-PEG-Ala-Val-ADM (yield from adriamycin: 44%).
  • the desired HO-PEG-Ala-Pro-ADM was eluted with 20 ml of a mixture of chloroform:methanol:water (13:6:1). The solvent was removed from the desired fraction under reduced pressure to give 182 ⁇ g (0.21 ⁇ mol) of HO-PEG-Ala-Pro-ADM (yield from adriamycin: 46%).
  • the desired HO-PEG-Gly-Pro-ADM was eluted with 20 ml of a mixture of chloroform:methanol:water (13:6:1). The solvent was removed from the desired fraction under reduced pressure to give 240 ⁇ g (0.19 ⁇ mol) of HO-PEG-Gly-Pro-ADM (yield from adriamycin: 62%).
  • the desired HO-PEG-Ala-Val-DNR was eluted with 20 ml of a mixture of chloroform:methanol:water (13:6:1). The solvent was removed from the desired fraction under reduced pressure to give 38 ⁇ g (0.03 ⁇ mol) of HO-PEG-Ala-Val-DNR (yield from daunorubicin: 1.8%).
  • the by-product, BzlO-PEG-Ala-Pro-DNR was removed by elution using, as the developer, 10 ml each of chloroform-methanol mixtures (10:1, 7:1, 5:1, 3:1, 2:1). Then, the desired HO-PEG-Ala-Pro-DNR was eluted with 20 ml of a mixture of chloroform:methanol:water (13:6:1). The solvent was removed from the desired fraction under reduced pressure to give 150 ⁇ g (0.1 ⁇ mol) of HO-PEG-Ala-Pro-DNR (yield from daunorubicin: 20%).
  • the by-product, BzlO-PEG-Gly-Pro-DNR was removed by elution using, as the developer, 10 ml each of chloroform-methanol mixtures (10:1, 7:1, 5:1, 3:1, 2:1). Then, the desired HO-PEG-Gly-Pro-DNR was eluted with 20 ml of a mixture of chloroform:methanol:water (13:6:1). The solvent was removed from the desired fraction under reduced pressure to give 180 ⁇ g (0.1 ⁇ mol) of HO-PEG-Gly-Pro-DNR (yield from daunorubicin: 26%).
  • Compounds (2) to (9) and Compound (11) were confirmed by 1 H-NMR and mass spectrometric analysis.
  • 20 mg (36.6 ⁇ mol) of Compound (11) was dissolved in 1.1 ml of a mixture of acetic acid (0.2 ml) and tetrahydrofuran (9.8 ml), and 7.3 mg of 10% palladium carbon catalyst was added thereto at 10°C to 15°C, followed by vigorous stirring in a stream of hydrogen at 10°C to 15°C for 3 hours and 20 minutes.
  • the resulting mixture was added under ice cooling to a solution prepared by adding 11.5 ml of a phosphate buffer to 8 ml of an aqueous solution of KM-641 antibody (1.47 mg/ml), followed by gentle stirring at 4°C for 24 hours. After the insoluble matter was removed with a filter (0.22 ⁇ m), the antibody fraction was purified by gel filtration chromatography [column: 200 ml of Sephacryl S 200 (Pharmacia Co., Ltd.), developer: a phosphate buffer, flow rate: 0.5 ml/minute, 11.5 ml fractions].
  • the 8th and 9th fractions were collected to obtain a solution containing 0.34 mg/ml KM-641-(PEG-Ala-Val-Segment B) m .
  • the number of molecules of Segment B bound per antibody molecule was calculated by subjecting the conjugate to enzyme treatment (thermolysin) and quantitatively determining released H-Val-Segment B by HPLC according to the method described in Reference Example 22. It was found that in the obtained conjugate, the number of molecules of Segment B was 1.9 per antibody molecule. It was confirmed that the affinity of the conjugate was approximately equal to that of an unbound antibody according to the following enzyme-linked immunosorbent assay.
  • Ganglioside GD 3 (2 nmol) was dissolved in 2 ml of ethanol containing 5 ng of phosphatidyl choline (Sigma Chemical Co.) and 2.5 ng of cholesterol, and the solution was put into wells of a 96-well plate for ELISA (Linbro Co., Ltd.) in an amount of 20 ⁇ l/well. After drying the wells, a phosphate buffer containing 1% bovine serum albumin was added to the wells for blocking. The above-described conjugate (10 ⁇ g/ml, 50 ⁇ l) was added to each well, and the plate was allowed to stand at room temperature for 2 hours (or at 4°C for 24 hours).
  • peroxidase-labelled rabbit anti-mouse Ig antibody (Dako) was added to the wells as the second antibody, and the plate was allowed to stand at room temperature for 1 to 2 hours, followed by washing.
  • ABTS Sigma Chemical Co.
  • spectrophotometry was carried out on the absorbance at 414 nm using NJ-2001 (Japan Intermed Co., Ltd.).
  • This Reference Example demonstrates that the peptide bond of a spacer bound to an antitumor agent is cleaved in a cell by a specific enzyme and that the cleavage of the spacer does not occur in a serum, using Compound (25) obtained in Reference Example 19.
  • the release of Segment B by the use of thermolysin as the cleavage enzyme was confirmed in the following manner. To 0.1 ml of a solution of Compound (25) in a phosphate buffer (0.2 mg/ml) was added 0.1 ml of an enzyme solution (0.1 mg/ml) (amount of enzyme: 94 pU), and the mixture was allowed to stand at 37°C for 24 hours.
  • This Reference Example demonstrates that the peptide bond of a conjugate of an antibody and an antitumor agent through a spacer is cleaved in a cell by a specific enzyme, but is stable in a serum, using the conjugate of an antibody and Segment B through a spacer obtained in Reference Example 20.
  • the specific cleavage of the spacer was confirmed as follows using thermolysin as the intracellular cleavage enzyme and plasmin as the main proteolytic enzyme in blood.
  • thermolysin 0.1 mg/ml
  • amount of enzyme: 2.4 pU 2.5 ⁇ l of a 1:200 dilution of plasmin (amount of enzyme: 250 ⁇ U)
  • amount of enzyme: 250 ⁇ U 5.7 ⁇ l of a phosphate buffer
  • the resulting supernatant was analyzed by reversed-phase HPLC under the same conditions as in Reference Example 21.
  • Reference Example 25 Enzyme-specific Cleavage of a Spacer Experiment using a spacer bound to adriamycin
  • Reference Example 26 Enzyme-specific Cleavage of a Spacer Experiment using a conjugate of an antibody and adriamycin through a spacer
  • Example 3 To 35 ⁇ l of Compound (Ia-3) obtained in Example 3 (0.19 mg/ml) were added 70 ⁇ l of proline endopeptidase (1.0 mg/ml) (amount of enzyme: 2.4 U) and 95 ⁇ l of a phosphate buffer, and the mixture was allowed to stand at 37°C for 24 hours. The resulting supernatant was analyzed by reversed-phase HPLC under the same conditions as in Reference Example 25, whereby the release of ADM from Compound (Ia-3) was confirmed (cleavage efficiency: 10%). Elution time: 23.7 minutes (agreed with that for ADM)
  • Example 2 To 40 ⁇ l of Compound (Ia-2) obtained in Example 2 (0.27 mg/ml) were added 110 ⁇ l of proline endopeptidase (1.0 mg/ml) (amount of enzyme: 3.9 U) and 50 ⁇ l of a phosphate buffer, and the mixture was allowed to stand at 37°C for 24 hours. The resulting supernatant was analyzed by reversed-phase HPLC under the same conditions as in Reference Example 25, whereby the release of ADM from Compound (Ia-2) was confirmed (cleavage efficiency: 10%). Elution time: 23.9 minutes (agreed with that for ADM)
  • Example 9 To 17 ⁇ l of Compound (Ia-9) obtained in Example 9 (1.2 mg/ml) were added 80 ⁇ l of proline endopeptidase (1.0 mg/ml) (amount of enzyme: 2.8 U) and 53 ⁇ l of a phosphate buffer, and the mixture was allowed to stand at 37°C for 24 hours. The resulting supernatant was analyzed by reversed-phase HPLC, whereby the release of Compound (20) from Compound (Ia-9) was confirmed. Reversed-phase HPLC conditions; The same apparatus and column as in Reference Example 21 were used.
  • Example 7 To 18 ⁇ l of Compound (Ia-7) obtained in Example 7 (1.1 mg/ml) were added 60 ⁇ l of thermolysin (3.0 mg/ml) (amount of enzyme: 1.7 ⁇ U) and 72 ⁇ l of a phosphate buffer, and the mixture was allowed to stand at 37°C for 24 hours. The resulting supernatant was analyzed by reversed-phase HPLC under the same conditions as in (1), whereby the release of H-Val-Compound (20) from Compound (Ia-7) was confirmed. Elution time: 17.7 minutes
  • Example 8 To 11 ⁇ l of Compound (Ia-8) obtained in Example 8 (1.8 mg/ml) were added 80 ⁇ l of proline endopeptidase (1.0 mg/ml) (amount of enzyme: 2.8 U) and 59 ⁇ l of a phosphate buffer, and the mixture was allowed to stand at 37°C for 24 hours. The resulting supernatant was analyzed by reversed-phase HPLC under the same conditions as in (1), whereby the release of Compound (20) from Compound (Ia-8) was confirmed. Elution time: 25.9 minutes [agreed with that for Compound (20)]
  • a cytotoxin conjugate which is useful as an active ingredient of an antitumor agent and an in vitro diagnosis technique using the conjugate is described.
  • the conjugate comprises a cytotoxin and a compound having an affinity for a target cell, for example, an antibody or antibody fragment which is specific to a cancer, said toxin and compound being bound through a spacer.

Landscapes

  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Claims (9)

  1. Conjugué de cytotoxine, dans lequel un résidu dérivé d'un composé qui possède une affinité pour une cellule cible est lié à une cytotoxine par l'intermédiaire d'un bras espaceur comprenant un polyalkylèneglycol et un dipeptide.
  2. Conjugué de cytotoxine représenté par la formule (I) :

            Z-(-X1-CH2(OCH2CH2)nOCH2CO-R1-R2-W-Y1)m      (I)

    dans laquelle :
    - Z représente un résidu dérivé d'un composé qui possède une affinité pour une cellule cible ;
    - X1 représente un chaînon -CO- ou -S-, ou un groupe de formule
    Figure imgb0033
    - W représente une liaison simple ou un groupe de formule
    Figure imgb0034
    - Y1 représente une cytotoxine ;
    - R1 et R2 représentent des résidus d'acides aminés, qui peuvent être identiques ou différents l'un de l'autre ;
    - n représente un nombre entier qui vaut de 1 à 1000 ;
    - et m représente un nombre entier qui vaut de 1 à 100.
  3. Conjugué de cytotoxine conforme à la revendication 2, dans lequel le résidu représenté par Z comporte un groupe carbonyle CO ou un atome d'azote N, de soufre S ou d'oxygène O, et la cytotoxine représentée par Y1 comporte un atome d'azote N, de soufre S ou d'oxygène O, pourvu que, si X1 représente un chaînon -S-, le résidu Z soit lié au chaînon X1 par l'intermédiaire d'un groupe carbonyle CO, et si X1 représente un raccord autre qu'un chaînon -S-, le résidu Z soit lié au raccord X1 par l'intermédiaire d'un atome d'azote N, de soufre S ou d'oxygène O, et la cytotoxine Y1 est liée au raccord W ou au résidu R2 par l'intermédiaire d'un atome d'azote N, de soufre S ou d'oxygène O.
  4. Conjugué de cytotoxine conforme à la revendication 2 ou 3, dans lequel Z représente un résidu dérivé d'une protéine ou d'un peptide.
  5. Conjugué de cytotoxine conforme à la revendication 4, dans lequel la protéine est un anticorps ou un fragment d'anticorps.
  6. Conjugué de cytotoxine conforme à l'une des revendications 2 à 5, dans lequel R1 représente un résidu d'alanine, de leucine ou de glycine, et R2 représente un résidu de proline, de valine ou de leucine.
  7. Conjugué de cytotoxine conforme à l'une des revendications 2 à 6, dans lequel Y1 représente un résidu dérivé de l'adriamycine, de la daunorubicine, d'un dérivé de duocarmycine, de la mitomycine A, de la mitomycine C, de la ricine A, de la toxine diphtérique ou d'une exotoxine de Pseudomonas.
  8. Conjugué de cytotoxine conforme à l'une des revendications 1 à 7, conçu pour servir dans le traitement d'un cancer.
  9. Emploi d'un conjugué de cytotoxine, conforme à l'une des revendications 1 à 7, dans la fabrication d'une composition conçue pour le traitement d'un cancer.
EP96913722A 1995-05-10 1996-05-10 Conjugues des cytotoxines comprenant des dipeptides Expired - Lifetime EP0867190B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP111933/95 1995-05-10
JP11193395 1995-05-10
PCT/JP1996/001241 WO1996035451A1 (fr) 1995-05-10 1996-05-10 Nouveau complexe de toxines

Publications (3)

Publication Number Publication Date
EP0867190A1 EP0867190A1 (fr) 1998-09-30
EP0867190A4 EP0867190A4 (fr) 2002-01-02
EP0867190B1 true EP0867190B1 (fr) 2007-12-26

Family

ID=14573780

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96913722A Expired - Lifetime EP0867190B1 (fr) 1995-05-10 1996-05-10 Conjugues des cytotoxines comprenant des dipeptides

Country Status (8)

Country Link
US (2) US6103236A (fr)
EP (1) EP0867190B1 (fr)
JP (1) JP3871713B2 (fr)
AT (1) ATE381948T1 (fr)
CA (1) CA2220339C (fr)
DE (1) DE69637383T2 (fr)
ES (1) ES2299177T3 (fr)
WO (1) WO1996035451A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8034959B2 (en) 2001-05-31 2011-10-11 Medarex, Inc. Methods of treating cancer with an antibody-drug conjugate
US8399403B2 (en) 2004-05-19 2013-03-19 Medarex, Inc. Chemical linkers and conjugates thereof

Families Citing this family (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6448369B1 (en) 1997-11-06 2002-09-10 Shearwater Corporation Heterobifunctional poly(ethylene glycol) derivatives and methods for their preparation
US7642323B2 (en) 1997-11-06 2010-01-05 Nektar Therapeutics Heterobifunctional poly(ethylene glycol) derivatives and methods for their preparation
MXPA00011312A (es) * 1998-05-20 2003-04-22 Expression Genetics Inc Un vehiculo de gen polimerico de poli-l.lisina injertada con polietilenglicol con porcion de enfoque hepatocitos.
JP4216480B2 (ja) * 1999-05-17 2009-01-28 コンジュケム バイオテクノロジーズ インコーポレイテッド ウイルス感染の長期持続性融合ペプチドインヒビター
US6258133B1 (en) * 1999-06-02 2001-07-10 Chevron Chemical Company Llc Poly (oxyalkylene) pyridyl and piperidyl ethers and fuel compositions containing the same
US6706892B1 (en) 1999-09-07 2004-03-16 Conjuchem, Inc. Pulmonary delivery for bioconjugation
US7090851B1 (en) 1999-09-10 2006-08-15 Conjuchem Inc. Long lasting fusion peptide inhibitors of viral infection
ATE388167T1 (de) 1999-09-30 2008-03-15 Kyowa Hakko Kogyo Kk Menschlicher antikörper gegen gangliosid gd3 für die transplantationskomplementarität bestimmente regionund derivate des antikörpers gegen das gangliosid gd3
CN1284603C (zh) * 1999-10-08 2006-11-15 内科塔治疗亚拉巴马公司 杂双官能聚乙二醇衍生物和它们的制备方法
AU5257401A (en) * 2000-04-28 2001-11-12 Asahi Chemical Ind Novel bicyclic compounds
US20040136908A1 (en) * 2001-04-09 2004-07-15 Olson William C. Anti-cd19 immunotoxins
US7741453B2 (en) * 2001-05-31 2010-06-22 Conjuchem Biotechnologies, Inc. Long lasting fusion peptide inhibitors for HIV infection
JP2008505059A (ja) * 2004-05-06 2008-02-21 コンジュシェム バイオテクノロジーズ インコーポレイティド 特異的ウイルス標的用化合物
JP4806680B2 (ja) * 2004-05-19 2011-11-02 メダレックス インコーポレイテッド 自己犠牲リンカー及び薬剤複合体
CN1997402B (zh) * 2004-05-19 2014-04-02 梅达雷克斯有限责任公司 化学连接基团及其缀合物
EP1693458A1 (fr) * 2005-02-17 2006-08-23 Universite Pierre Et Marie Curie Peptides inhibiteurs intracellulaires
US7714016B2 (en) * 2005-04-08 2010-05-11 Medarex, Inc. Cytotoxic compounds and conjugates with cleavable substrates
BRPI0617546A2 (pt) * 2005-09-26 2011-07-26 Medarex Inc conjugado de fÁrmaco-anticorpo, formulaÇço farmacÊutica, mÉtodo para matar uma cÉlula de tumor, mÉtodo para retardar ou interromper o crescimento de um tumor em um sujeito mamÍfero e composto
SI1940789T1 (sl) 2005-10-26 2012-03-30 Medarex Inc Postopki in spojine za pripravo cc analogov
CA2627190A1 (fr) 2005-11-10 2007-05-24 Medarex, Inc. Composes et conjugues cytotoxiques
KR101513732B1 (ko) 2006-02-21 2015-04-21 넥타르 테라퓨틱스 분할된 분해가능한 폴리머 및 이로부터 제조된 컨주게이트
MX2009002418A (es) 2006-09-05 2009-04-23 Medarex Inc Anticuerpos para las proteinas morfogenicas oseas y receptores de estas y metodos para su uso.
TWI412367B (zh) 2006-12-28 2013-10-21 梅達雷克斯有限責任公司 化學鏈接劑與可裂解基質以及其之綴合物
US20090088378A1 (en) * 2007-01-12 2009-04-02 Omar Quraishi Long lasting inhibitors of viral infection
CA2678514A1 (fr) 2007-02-21 2008-08-28 Medarex, Inc. Liants chimiques avec acides amines uniques et conjugues de ceux-ci
CN101687911A (zh) * 2007-05-16 2010-03-31 康久化学生物技术公司 抗病毒肽的半胱磺酸衍生物
EP2214716B1 (fr) 2007-10-23 2021-11-17 Nektar Therapeutics Polymères branchés ciblant l'hydroxyapatite et conjugués préparés à partir de ces polymères
US9238878B2 (en) 2009-02-17 2016-01-19 Redwood Bioscience, Inc. Aldehyde-tagged protein-based drug carriers and methods of use
JP5767207B2 (ja) 2010-03-26 2015-08-19 協和発酵キリン株式会社 新規修飾部位導入抗体および抗体フラグメント
AU2012205301B2 (en) 2011-01-14 2017-01-05 Redwood Bioscience, Inc. Aldehyde-tagged immunoglobulin polypeptides and method of use thereof
US10131682B2 (en) 2012-11-24 2018-11-20 Hangzhou Dac Biotech Co., Ltd. Hydrophilic linkers and their uses for conjugation of drugs to a cell binding molecules
WO2014089177A2 (fr) 2012-12-04 2014-06-12 Massachusetts Institute Of Technology Composés, conjugués et compositions d'épipolythiodicétopipérazines et de polythiodicétopipérazines
PL2968440T3 (pl) 2013-03-15 2019-12-31 Zymeworks Inc. Związki cytotoksyczne i antymitotyczne oraz sposoby ich stosowania
RU2729194C2 (ru) 2013-12-27 2020-08-05 Займворкс Инк. Сульфонамидсодержащие связывающие системы для лекарственных конъюгатов
CA2938919C (fr) 2014-02-28 2020-12-29 Hangzhou Dac Biotech Co., Ltd Lieurs charges et leurs utilisations pour la conjugaison
AU2015318556C1 (en) 2014-09-17 2021-01-07 Zymeworks Bc Inc. Cytotoxic and anti-mitotic compounds, and methods of using the same
FI3319936T3 (fi) 2015-07-12 2026-03-12 Hangzhou Dac Biotech Co Ltd Silloituslinkkereitä soluun sitoutuvien molekyylien konjugoimiseksi
US9839687B2 (en) 2015-07-15 2017-12-12 Suzhou M-Conj Biotech Co., Ltd. Acetylenedicarboxyl linkers and their uses in specific conjugation of a cell-binding molecule
AU2017257504A1 (en) 2016-04-26 2018-10-25 R.P. Scherer Technologies, Llc Antibody conjugates and methods of making and using the same
US10918627B2 (en) 2016-05-11 2021-02-16 Massachusetts Institute Of Technology Convergent and enantioselective total synthesis of Communesin analogs
CA3042442C (fr) 2016-11-14 2024-01-02 Hangzhou Dac Biotech Co., Ltd Lieur de conjugaison, conjugues molecule-medicament de liaison de cellules contenant les lieurs, methodes de production et utilisations detels conjugues avec les lieurs
WO2018209239A1 (fr) 2017-05-11 2018-11-15 Massachusetts Institute Of Technology Dérivés d'agélastatine puissants en tant que modulateurs de l'invasion et de la métastase du cancer
US10640508B2 (en) 2017-10-13 2020-05-05 Massachusetts Institute Of Technology Diazene directed modular synthesis of compounds with quaternary carbon centers
US11535634B2 (en) 2019-06-05 2022-12-27 Massachusetts Institute Of Technology Compounds, conjugates, and compositions of epipolythiodiketopiperazines and polythiodiketopiperazines and uses thereof
EP4141017A4 (fr) 2020-04-24 2024-05-08 The University of Tokyo Dérivé de duocarmycine et son utilisation
WO2022182415A1 (fr) 2021-02-24 2022-09-01 Massachusetts Institute Of Technology Dérivés d'himastatine, leurs procédés de préparation et leurs utilisations
CN117980327A (zh) 2021-11-03 2024-05-03 杭州多禧生物科技有限公司 抗体的特异性偶联

Family Cites Families (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
YU33730B (en) * 1967-04-18 1978-02-28 Farmaceutici Italia Process for preparing a novel antibiotic substance and salts thereof
US4671958A (en) * 1982-03-09 1987-06-09 Cytogen Corporation Antibody conjugates for the delivery of compounds to target sites
CA1203164A (fr) * 1982-03-09 1986-04-15 Thomas J. Mckearn Conjugats d'anticorps
JPS6067433A (ja) 1983-09-24 1985-04-17 Kyowa Hakko Kogyo Co Ltd マイトマイシン類，多糖類及び抗体の複合体
US4732863A (en) * 1984-12-31 1988-03-22 University Of New Mexico PEG-modified antibody with reduced affinity for cell surface Fc receptors
JPS6335575A (ja) 1986-07-31 1988-02-16 Kyowa Hakko Kogyo Co Ltd マイトマイシン誘導体
JPS63246336A (ja) 1986-11-10 1988-10-13 Kyowa Hakko Kogyo Co Ltd マイトマイシン−抗体複合体含有組成物
JPS63150282A (ja) * 1986-12-13 1988-06-22 Kyowa Hakko Kogyo Co Ltd マイトマイシン誘導体
JPS63256336A (ja) * 1987-04-13 1988-10-24 Yoshiaki Kakino Ｎｃ機械におけるボ−ルねじの熱変位補正方法
US5219564A (en) * 1990-07-06 1993-06-15 Enzon, Inc. Poly(alkylene oxide) amino acid copolymers and drug carriers and charged copolymers based thereon
US5169934A (en) * 1990-05-14 1992-12-08 Anergen, Inc. Intracellularly cleavable compounds
DK0610179T3 (da) * 1990-07-20 1997-03-24 Pharmacia & Upjohn Ab Målspecifikke antistof-superantigenkonjugater og fremstilling deraf
DK0575545T3 (da) * 1991-03-15 2003-09-15 Amgen Inc Pegylering af polypeptider
JPH07500315A (ja) * 1991-05-10 1995-01-12 セルトリックス　ファーマシューティカルズ，インコーポレイテッド 骨成長因子の標的送達
US5169627A (en) * 1991-10-28 1992-12-08 Mount Sinai School Of Medicine Of The City University Of New York Oral pharmaceutical composition containing a polyethylene glycol-immunoglobulin G conjugate for reconstitution of secretory immunity and method of reconstituting secretory immunity
EP0571330B1 (fr) * 1992-05-22 1999-04-07 Ciba SC Holding AG Photoréserve à haute résolution et à sensibilité augmentée pour l'exposition avec des lampes émettant en lignes I
JPH06335575A (ja) * 1993-05-28 1994-12-06 Tokyo Electric Co Ltd 電気かみそり

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8034959B2 (en) 2001-05-31 2011-10-11 Medarex, Inc. Methods of treating cancer with an antibody-drug conjugate
US8399403B2 (en) 2004-05-19 2013-03-19 Medarex, Inc. Chemical linkers and conjugates thereof

Also Published As

Publication number Publication date
CA2220339A1 (fr) 1996-11-14
JP3871713B2 (ja) 2007-01-24
ATE381948T1 (de) 2008-01-15
DE69637383D1 (de) 2008-02-07
US6638509B1 (en) 2003-10-28
CA2220339C (fr) 2009-10-13
WO1996035451A1 (fr) 1996-11-14
DE69637383T2 (de) 2008-12-11
ES2299177T3 (es) 2008-05-16
EP0867190A1 (fr) 1998-09-30
EP0867190A4 (fr) 2002-01-02
US6103236A (en) 2000-08-15

Similar Documents

Publication Publication Date Title
EP0867190B1 (fr) Conjugues des cytotoxines comprenant des dipeptides
JPWO1996035451A1 (ja) 新規毒素複合体
CA1243015A (fr) Agent de liberation dans les cellules cibles
KR100281606B1 (ko) 중합체-결합 된파클리탁셀 유도체, 이의 제조방법 및 이를 포함하는 약제학적 조성물
US6410514B1 (en) Tissue specific prodrug
US5037883A (en) Synthetic polymeric drugs
US7767648B2 (en) Tissue specific prodrugs
JP2001505194A (ja) 分枝ペプチド・リンカー
JP2008519863A (ja) Ｎ末端にアミノ安息香酸単位を有するオーリスタチン
HU208161B (en) Process for producing cytotoxic active ingredient - antibody conjugates and pharmaceutical compositions
WO2024012569A9 (fr) Lieurs, conjugués et leurs applications
EP3335734A1 (fr) Lieur-médicament et conjugué anticorps-médicament (adc) l'utilisant
Suzawa et al. Synthesis of a novel duocarmycin derivative DU-257 and its application to immunoconjugate using poly (ethylene glycol)-dipeptidyl linker capable of tumor specific activation
JP2008531617A (ja) 蛋白質結合性アントラサイクリンペプチド誘導体、およびそれを含む医薬
RU2439077C2 (ru) Белоксвязывающие производные метотрексата и содержащие их лекарства
US5272253A (en) Cluster conjugates of drugs with antibodies
EP4578464A1 (fr) Composition pharmaceutique de conjugué anticorps-médicament
Suzawa et al. Synthesis and HPLC analysis of enzymatically cleavable linker consisting of poly (ethylene glycol) and dipeptide for the development of immunoconjugate
WO2025140358A1 (fr) Composition pharmaceutique de conjugué anticorps-médicament anti-fgfr3 et ses applications
RU2798981C2 (ru) Конъюгат антитела с аматоксином неприродного типа
EP4483907A1 (fr) Nouveau conjugué anticorps-médicament
RU2840926C2 (ru) Конъюгат антитела с аматоксином неприродного типа
EP0489220A1 (fr) Dérivés N,N-bis(succinyl-peptidiques) cytotoxiques d'1,4-bis(aminoalkyl)-5,8-dihydroxyanthraquinones et leurs conjuqués d'anticorps
HK40035180A (en) Non-natural amatoxin-type antibody conjugate

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19971208

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

RIN1 Information on inventor provided before grant (corrected)

Inventor name: HANAI, NOBUO

Inventor name: OHTA, SO

Inventor name: SAITO, HIROMITSU

Inventor name: NAGAMURA, SATORU

Inventor name: YAMASAKI, MOTOO

Inventor name: SUZAWA, TOSHIYUKI

A4 Supplementary search report drawn up and despatched

Effective date: 20011120

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

RIC1 Information provided on ipc code assigned before grant

Free format text: 7A 61K 47/48 A

17Q First examination report despatched

Effective date: 20050321

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RTI1 Title (correction)

Free format text: CYTOTOXIN COMPLEXES COMPRISING DIPEPTIDES

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REF Corresponds to:

Ref document number: 69637383

Country of ref document: DE

Date of ref document: 20080207

Kind code of ref document: P

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080326

Ref country code: LI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20071226

Ref country code: CH

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20071226

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2299177

Country of ref document: ES

Kind code of ref document: T3

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20071226

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20071226

NLV1 Nl: lapsed or annulled due to failure to fulfill the requirements of art. 29p and 29m of the patents act
REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20071226

ET Fr: translation filed
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20071226

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080526

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20071226

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20080929

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080531

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080327

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080512

REG Reference to a national code

Ref country code: FR

Ref legal event code: CD

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080510

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20110523

Year of fee payment: 16

Ref country code: ES

Payment date: 20110520

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20110504

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20110505

Year of fee payment: 16

Ref country code: IT

Payment date: 20110519

Year of fee payment: 16

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20120510

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120510

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20130131

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 69637383

Country of ref document: DE

Effective date: 20121201

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120531

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120510

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20121201

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20130820

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120511